KR20220109589A - Pharmatheutical composition comprising miR-486-5p for preventing or treating of hepatic fibrosis - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis comprising miR-486-5p, an activator thereof or an expression promoter thereof as an active ingredient. More specifically, since miR-486-5p inhibits the activation of hepatic stellate cells (HSC) by inhibiting the expression of SMO, a receptor in the Hedgehog (Hh) pathway, it is possible to be usefully used in the prevention or treatment of liver fibrosis by suppressing the progression of liver fibrosis.

Description

A pharmaceutical composition for preventing or treating liver fibrosis comprising miR-486-5p as an active ingredient TECHNICAL FIELD

The present invention relates to a pharmaceutical composition for preventing or treating liver fibrosis comprising miR-486-5p or an activator thereof or an expression promoter thereof as an active ingredient, specifically miR-486-5p is Hedgehog (Hh) By suppressing the expression of the receptor SMO in the pathway to inhibit the activation of hepatic stellate cells (HSC), it can be usefully used in the prevention or treatment of liver fibrosis by inhibiting the progression of hepatic fibrosis.

Chronic liver disease develops in response to persistent long-term liver damage, such as viral hepatitis, obesity, or alcohol abuse. Although the causes of liver disease may vary, the common pathological feature of most chronic liver diseases is liver fibrosis. Hepatic fibrosis, which fills the area that cannot be regenerated by hepatocytes with extracellular matrix (ECM), leads to excessive accumulation of extracellular matrix (ECM) due to chronic and/or severe liver damage, which eventually disrupts the normal function of the liver. It leads to the terminal stages of cirrhosis or liver cancer. Hepatic stellate cells (HSC), which are the main cause of hepatic fibrosis, are a type of fibrogenic cell, and liver fibrosis is initiated by activation of hepatic stellate cells (HSC). In normal liver, hepatic stellate cells (HSCs), which are in a resting state (Q-), maintain a nonproliferative phenotype and store vitamin A/retinoic acid. During liver injury, hepatic stellate cells (HSCs) are activated and proliferate in the damaged liver, and are transformed into myofibroblasts and hepatocytes that produce collagen-like extracellular matrix (ECM) and differentiated. Therefore, modulating hepatic stellate cell (HSC) activation could be a potential therapeutic strategy for anti-fibrosis therapy.

On the other hand, microRNA (microRNA) is a small non-protein-coding RNA that regulates a wide range of biological pathways, such as cell proliferation, differentiation and apoptosis, by controlling the expression of hundreds of genes through RNA interference in the post-transcriptional process of genes ( Calin et al., Nat. Rev. Cancer, 6: 857-866 (2006)). Although microRNAs are useful for predicting or treating the clinical prognosis of various diseases, there have been few reports of microRNAs effective for preventing or treating liver fibrosis yet.

Under this background, the present inventors tried to discover microRNAs effective for preventing or treating liver fibrosis.

Korean Patent Registration No. 10-1835018 (Registered on February 27, 2018)

The inventors of the present invention evaluated the therapeutic effect of tonsil-derived mesenchymal stem cells (T-MSC) on exosome-derived miR-486-5p on liver fibrosis, and miR-486-5p significantly increased in liver fibrosis animal models. The present invention was completed by confirming that there is an excellent liver fibrosis treatment effect.

Accordingly, an object of the present invention is a pharmaceutical composition for preventing or treating liver fibrosis comprising miR-486-5p, an activator thereof or an expression promoter thereof as an active ingredient. Or to provide a health functional food composition for improving liver liver fibrosis.

Therefore, in order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, comprising miR-486-5p, an activator thereof, or an expression promoter thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for improving liver fibrosis, comprising miR-486-5p, an activator thereof or an expression promoter thereof as an active ingredient.

The composition for preventing or treating liver fibrosis according to the present invention suppresses the expression of SMO, a receptor, in the Hedgehog pathway, thereby inhibiting the activation of Hepatic stellate cells (HSC), and collagen deposition in the liver ( collagen deposition), it can be usefully used for the prevention or treatment of liver fibrosis.

1 is a psiCHECK-2 vector with a wild-type or mutant binding site of miR-486-5p at the 3' UTR of SMO mRNA. Wild-type (WT) or mutant (MUT) nucleotides of the miR-486-5p binding site on the 3'UTR of SMO mRNA are indicated. Mutated nucleotides are underlined.
2 is a result confirming that miR-486-5p directly targets SMO. (A) The potential binding site (red) of miR-486-5p was predicted in the 3'-UTR of mouse and human SMO mRNA using the miRNA database (miRWalk). The dotted line indicates the complementary base pairing between miR-486-5p and SMO mRNA, and the gray shade indicates the seed sequence of miR-486-5p. (B) Double luciferase analysis was performed to confirm the binding interaction between miR-486-5p and SMO mRNA. HepG2 or LX2 was co-transfected with a psiCHECK-2 vector containing a wild-type (wt) or mutant (mut) target site and either a miR-486-5p mimic (mimic) or a scrambled (Scr-)miR (control) infected. Results of relative luciferase assay activity were expressed as mean ± sem across three experiments (Student's test for two unpaired samples, *p <0.05, **p <0.005 vs. self-control).
3 is a result confirming that MiR-486-5p reduces the expression of HSC activation marker and Hh activation gene. (A) miR-486-5p, SMO in pHSCs (primary hepatic astrocytes isolated from human liver) transfected with Scr-miR or miR-486-5p mimetics for 1 day (D1) or 2 days (D2) , are the results of QRT-PCR analysis of GLI2, α-SMA, VIMENTIN, CTGF and TGF-β. Results from triplicate experiments are presented as mean ± sem (Student's t test for two unpaired samples, *p <0.05, **p <0.005 vs. self-control). (B) Western blot results. (C) Cumulative densitometry analysis results of SMO (86 kDa), GLI2 (133 kDa), TGF-β (25 kDa), α-SMA (42 kDa), VIMENTIN (57 kDa) and GAPDH (36 kD). GAPDH was used as an internal control, and the indicated immunoblots represent one of three independent experiments with similar results. The band density was normalized to the expression level of GAPDH used as an internal control. Results from triplicate experiments are presented as mean ± sem (Student's t test for two unpaired samples, *p <0.05, **p <0.005 vs. self-control).
4 is a result confirming that miR-486-5p alleviates CCl ₄ induced acute liver injury by targeting Smo. (A) Representative images of H&E-stained liver sections from representative mice of the CON+Scr group, CON+miR-486-5p group, CCl ₄ +Scr group, and CCl ₄ +miR-486-5p group (×10, Scale bar = 100 μm). (B) Results of measuring the serum levels of AST and ALT in this group (n = 3 per group). (C) The results of QRT-PCR analysis of G6pc in the liver of each group (n = 3 per group). (D) QRT-PCR analysis of miR-486-5p, Smo, Gli2, α-Sma, Vimentin, Ctgf and Col1α1 in mice (n = 3 per group). All statistical data are expressed as mean ± sem. (Student's t test for two unpaired samples, *p <0.05, **p <0.005). (E) Representative images of Sirius red staining (top) and immunostaining for α-Sma (bottom) in liver sections of this group (×10, scale bar = 100 μm).

The present inventors found that miR-486-5p inhibits the expression of SMO, a receptor, in the Hedgehog pathway, thereby inhibiting the activation of Hepatic stellate cells (HSC), preventing or treating liver fibrosis. The present invention was completed by finding that it can be usefully used for

Hereinafter, the present invention will be described in more detail.

The present invention provides a pharmaceutical composition for preventing or treating liver fibrosis, comprising miR-486-5p, an activator thereof, or an expression promoter thereof as an active ingredient.

Specifically, the miR-486-5p may be composed of SEQ ID NO: 1 below.

microRNA base sequence SEQ ID NO: miR-486-5p 3’-GAGCCCCGUCGAGUCAUGUCCU-5’ One

As used herein, the term “hepatic fibrosis” refers to a condition in which liver fibers are increased as a result of liver disorders.

The liver fibrosis preferably includes both viral liver fibrosis (HBV infection) and non-viral liver fibrosis, and more preferably non-viral liver fibrosis, but is not limited thereto. More specifically, the non-viral liver fibrosis is liver fibrosis, alcoholic liver cirrhosis, non-alcoholic cirrhosis, autoimmune cirrhosis, primary biliary cholangitis-associated cirrhosis, Wilson disease-associated cirrhosis, hemochromatosis-associated cirrhosis, portal cirrhosis, post-necrotic cirrhosis, It is more preferable that any one or more diseases selected from the group consisting of cirrhosis of the liver and cardiac cirrhosis associated with malnutrition are more preferable, but the present invention is not limited thereto.

As used herein, the term "liver fibrosis" refers to collagen and extracellular matrix (ECM) secreted in large amounts from active hepatic stellate cells as the inflammatory reaction continues due to tissue damage. ) refers to the process of wound healing that appears by combining If hepatic fibrosis continues, a large amount of collagen in the liver tissue is deposited, and the regenerated nodules are surrounded by collagen and can develop into cirrhosis, which has an abnormal structure.

In one embodiment of the present invention, the miR-486-5p inhibits the expression of SMO, a receptor, in the Hedgehog pathway, thereby inhibiting the activation of hepatic stellate cells (HSC), thereby preventing liver fibrosis. can be suppressed, and this is specifically confirmed in the Examples of the present invention. It can also reduce collagen deposition in the liver.

In another embodiment of the present invention, the composition may reduce blood aspartate aminotransferase (AST), blood alanine aminotransferase (ALT), and LW/BW (Ratio of liver weight to body weight) levels. can be reduced

The composition of the present invention may further include one or more known active ingredients having a preventive or therapeutic effect on liver fibrosis.

The pharmaceutical composition of the present invention may be formulated and administered in a unit dosage form suitable for intra-body administration of an individual according to a conventional method in the pharmaceutical field. Formulations suitable for this purpose include, for parenteral administration, injections such as ampoules for injection, injections such as infusion bags, and sprays such as aerosol formulations. The injection ampoule may be prepared by mixing with an injection solution immediately before use, and physiological saline, glucose, Ringer's solution, etc. may be used as the injection solution. In addition, the injection bag may be made of polyvinyl chloride or polyethylene.

The composition of the present invention may further include an appropriate carrier commonly used in the preparation of pharmaceutical compositions. For example, in the case of an injection, a preservative, an analgesic agent, a solubilizer or a stabilizer, etc., and in the case of a formulation for topical administration, a base, an excipient, a lubricant or a preservative may be further included.

The composition of the present invention may be administered to an individual by various routes. Any mode of administration can be anticipated, for example, it can be administered by oral, rectal, intraperitoneal, intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection, preferably intraperitoneal administration. However, the present invention is not limited thereto.

The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the treatment or prevention of liver fibrosis.

In the present invention, the term “prevention” may refer to any act of inhibiting or delaying the onset of liver fibrosis by administering the composition for preventing or treating liver fibrosis according to the present invention to an individual.

In the present invention, the term “treatment” may refer to any act of administering the composition according to the present invention to a subject suspected of having liver fibrosis so that the symptoms of liver fibrosis are improved or beneficial. As used herein, the term "individual" may refer to any animal, including humans, that has or is likely to develop liver fibrosis.

In addition, the composition of the present invention may be added to a food composition, preferably a health functional food, for the purpose of preventing or improving liver fibrosis.

In the present invention, 'health functional food' refers to a food having bioregulatory functions such as prevention and improvement of disease, body defense, immunity, recovery from illness, and suppression of aging, and should be harmless to the body when taken for a long time.

The mixing amount of the active ingredient contained in the food composition of the present invention may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment), but is not limited thereto.

As mentioned above, the composition for preventing or treating liver fibrosis according to the present invention inhibits the expression of SMO, a receptor, in the hedgehog (Hh) pathway, thereby inhibiting the activation of Hepatic stellate cells (HSC) and , a bar that reduces collagen deposition in the liver, can be usefully used for the prevention or treatment of liver fibrosis.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

<Experimental example>

The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

1. Cell Experiments

LX2, human HSC line (provided by Korea Advanced Institute of Science and Technology), human primary HSC (pHSC, purchased from Zen-Bio Inc., NC, USA), HepG2, human hepatocellular carcinoma cell line 10 in Dulbecco's Modified Eagle Medium (DMEM; Gibco) Dulbecco (provided by Pusan National University) line with % FBS (Gibco) and 1% P/S (Gibco) was cultured at 37° C. in a humid atmosphere containing 5% CO ₂ .

To investigate the effect of miR-486-5p on HSC activation, pHSCs at 70% confluence were serum-starved overnight in FBS-free medium and cultured in 2% FBS and P/S-free medium for 24 h. . Then, 25 nM of miR-486-5p analog (Bioneer, Daejeon, Korea) or scrambled miRNA (Bioneer, Daejeon, Korea) as a negative control using Lipofectamine RNAiMAX transfection reagent (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's instructions ) to transfect the cells.

After 6 hours, the transfected cells were cultured for 24 or 48 hours after replacement with fresh medium containing 2% FBS and 1% P/S.

2. Animal Model Experiments

Male C57BL/6 mice were purchased from Hyochang (Dae-gu, Korea), housed on a 12 h light-dark cycle, and had free access to normal food and water.

To investigate the direct effect of miR-486-5p in mice with acute liver injury, 7-week-old male C57BL/6 mice received 1.0 mg/kg body weight of CCl ₄ (n = 10) intraperitoneally twice for 1 week. On day 8, miR-486-5p analog (n = 5) or scrambled miRNA (n = 5) 8 nmol per mouse was intraperitoneally injected using in vivo-jetPEI (Polyplus Transfection, Illkirch, France). As a control, mice injected with corn oil (n = 8) were treated with miR-486-5p mimics (n = 4) or scrambled miRNAs (n = 4). Mice were weighed at the time of sacrifice and blood samples were collected by sterile cardiac puncture under isoflurane inhalation anesthesia.

The mice were then humanely euthanized under anesthesia by cervical dislocation and the livers were immediately dissected from the animals. All animal care and surgical procedures were approved by the Institutional Animal Care and Use Committee of Pusan National University and were performed in accordance with the provisions of the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals.

3. RNA Analysis

Total RNAs (Total RNAs) were extracted from cells, liver tissue or exosomes using TRIzol reagent (Ambion, Thermo Fisher Scientific).

The purity and concentration of RNA were determined using nanodrops. Template cDNA was synthesized from total RNAs using SuperScript Ⅱ First-strand Synthesis System (Invitrogen) or HB_I RT Reaction kit (HeimBioteck, Seongnam, Korea) according to the manufacturer's instructions.

QRT-PCR (Mastercycler realplex Real-time PCR, Eppendorf, Hamburg, Germany) using Power SYBR Green Master Mix (Applied Biosystems, Thermo Fisher Scientific) or HB_I Real-Time PCR Master Mix (HeimBioteck) according to manufacturer's specifications (Mastercycler realplex Real-time PCR, Eppendorf, Hamburg, Germany). Quantitative real-time PCR) was performed. All reactions were performed in triplicate, and data were analyzed according to the ΔΔC _t method. For normalization of expression levels, 40S ribosomal protein S9 mRNA and U6B small nuclear RNA (RNU6B) were used for mRNA and miRNA, respectively. For the sequences of all primers used to evaluate mRNA expression, those commonly used were used. In addition, for miR-486-5p, commercially pre-designed primers were purchased from HeimBioteck. PCR products were directly sequenced for identification of all genes (Macrogen, Seoul, Korea).

4. Western blot analysis

Total protein was extracted from cells, liver tissue or exosomes using Triton lysis buffer (TLB) supplemented with a protease inhibitor (Complete Mini 11836153001; Roche, Indianapolis, IN, USA). Nuclear fractionation was performed for Gli2 detection in liver tissue. Protein concentration was measured with a Pierce BCA protein assay kit (Thermo Fisher Scientific).

The same amount of protein was separated by 8% or 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (PVDF) (Millipore, Darmstadt, Germany). The primary antibodies used in this study were: CD63 (dilution 1:200; sc-5275, Santacruz Biotech.), CD9 (dilution 1:500; ab92726, Abcam), CD81 (dilution 1:200; sc- 16602, Santacruz Biotech.), calreticulin (dilution 1:1000; ab2907, Abcam), TGF-β (dilution 1:1000; 3711S, Cell Signaling Technology, MA, USA), α-SMA (dilution 1:1000; A5228, Sigma-Aldrich ), VIMENTIN (dilution 1: 1000; sc-5565, Santacruz Biotech.), COL1α1 (dilution 1: 1000; NBP1-30054, Novus Biologicals, CO, USA), GFAP (dilution 1: 1000; Z0334, Dako) , Carpinteria, CA, USA), SMO (dilution 1:1000; ab72130, Abcam), GLI2 (dilution 1:1000; GWB-CE7858, Genway Biotech., CA, USA), LAMIN B1 (dilution 1:1000; ab16048, Abcam) and GAPDH (dilution 1:1000; MCA4739, AbD Serotec, Oxford, UK). Horseradish peroxidase (HRP)-conjugated anti-rabbit (ADI-SAB-300-J, ENZO Life science, NY, USA) and anti-mouse lgG (ADI-SAB-100-J, ENZO Life science) as secondary antibodies was used. In addition, protein bands were detected using the EzWestLumi ECL solution (ATTO Corporation, Tokyo, Japan) according to the manufacturer's specifications (Ez-Capture II, ATTO Corporation). Cumulative densitometric analysis of immunoblots was measured using CS Analyzer 4 software (ATTO Corporation).

5. Liver histology and immunohistochemistry

Liver specimens were fixed in 10% neutral buffered formalin (Sigma-Aldrich), embedded in paraffin, and cut into 4 μm thick sections. Sections were dewaxed, hydrated and stained according to standard protocols using standard H&E staining to examine morphology and Sirius Red staining for fibrosis processes. For H&E staining, sections were stained with hematoxylin (Gill's Hematoxylin V, Muto Pure Chemicals, Tokyo, Japan) for 15 minutes and then with eosin (1% Eosin Y Solution, Muto Pure Chemicals) for 5 minutes. For Sirius Red staining, sections were incubated for 30 min in 0.1% Sirius Red F3B (Sigma-Aldrich) containing saturated picric acid and 0.1% Fast Green.

For immunohistochemistry, sections were incubated in 3% hydrogen peroxide for 10 min to block endogenous peroxidase. The antigen was recovered by heating in 10 mM sodium citrate buffer (pH 6.0). After washing the sections with TBS, they were treated with Dako protein block (Dako) for 30 min, 4 together with primary antibody, anti-α-SMA antibody (ab5694; Abcam) and anti-GLI2 (GWB-CE7858; Genway Biotech). Incubated overnight at °C. In addition, to demonstrate staining specificity, additional sections were incubated overnight at 4°C in non-immune serum. HRP-conjugated anti-rabbit (K4003; Dako) was used as a secondary antibody. According to the detection procedure, 3,3'-diaminobenzidine (Dako) was used. Slides were observed with an Olympus CX41 optical microscope (Olympus Optical Co. Ltd, Tokyo, Japan), and morphological analysis of stained areas of tissue sections was performed using cellSens software (Olympus Optical Co. Ltd).

6. AST/ALT measurement

Serum aspartate aminotransferase (AST/GOT, glutamate-oxaloacetate transaminase) and alanine aminotransferase (ALT/GPT, glutamate pyruvate transaminase) were treated with GOT and GPT reagents (Asan Pharmaceutical, Seoul, Korea). was used and measured according to the manufacturer's instructions.

7. Cloning of Vector Constructs and Analysis of Luciferase Reporter

For cloning of the vector construct, the target gene of human miR-486-5p was predicted through bioinformatics analysis using an online database (http://mirwalk.umm.uni-heidelberg.de). The 3'-UTR of human SMO was amplified by PCR in genomic DNA containing a binding site for human miR-486-5p.

The primer sequences used for vector construction are as follows: Forward primer: TTT TCT CGA GGG GGC CAT GTC CTC TCT TAA (SEQ ID NO: 2), Reverse primer: TGC GGC CGC TTG GAG GCT ATG GAA GGT GG (SEQ ID NO: 3). PCR. The product was purified using AccuPrep PCR Purification Kit (Bioneer), digested with restriction enzymes Xho1 and Not1, and cloned into a psiCHECK2 vector (Promega, WI, USA).

The vector construct containing 3'-UTR of SMO was transformed into E. coli, and plasmid DNA was extracted from the transformed ampicillin-resistant E. coli using AccuPrep Plasmid Mini Extraction Kit (Bioneer). The miR-486-5p binding site of 3'-UTR of SMO was confirmed through sequencing analysis. A mutant vector without miR-486-5p binding site was prepared by Enzynomics (Daejeon, Korea).

For the luciferase reporter assay, HepG2 or LX2 cells were seeded into 24-well culture plates in culture medium without P/S one day prior to transfection. Lipofectamine 2000 (Invitrogen) was used to transfect the cells with the psiCHECK-2 vector construct and 25 nM miR-486-5p mimetic [5'-UCCUGUACUGAGCUGCCCCGAG-3' (SEQ ID NO: 4); Bioneer] or a mixture of scrambled miRNA (SMC-2003; Bioneer) as a negative control. Cells were harvested 48 hours after transfection and performed with the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's protocol. All firefly luciferase activity data were normalized to Renilla luciferase activity and expressed as mean ± s.e.m of values obtained from at least three replicates.

8. Statistical Analysis

Data results were expressed as mean±s.e.m, and statistical differences were analyzed by unpaired two-sample Student’s t-test. P-values <0.05 were considered statistically significant.

Example 1. Inhibition of hepatocyte activity through inhibition of SMO expression of miR-486-5p

Through bioinformatics analysis using miRWalk, smoothened (SMO), a receptor in the Hh pathway, was predicted as a putative target of miR-486-5p (Fig. 2A). Through luciferase reporter analysis, it was confirmed that miR-486-5p directly bound to the 3'-untranslated region (3'-UTR) of SMO mRNA in both HepG2 or LX2 cells (Fig. 2B).

Furthermore, to investigate whether miR-486-5p affects HSC activation, human activated pHSCs were transfected with miR-486-5p mimic (mimic) or scramble (Scr)-miR. Although miR-486-5p was hardly expressed in activated pHSCs, its expression was greatly increased in pHSCs transfected with miR-486-5p. Sequentially, it was confirmed that the expression levels of SMO and glioblastoma 2 (GLI2) targeting the Hh transcription factor miR-486-5p were significantly reduced compared to cells transfected with scramble (Scr)-miR (Fig. 3A). ).

In addition, compared to Scr-miR, pHSCs transfected with miR-486-5p had down-regulated profibrotic markers TGF-β, α-SMA, VIMENTIN and CTGF. Western blot analysis confirmed the reduction of SMO, GLI2 and profibrotic markers in pHSCs transfected with miR-486-5p ( FIGS. 3B and 3C ). These results suggest that miR-486-5p inhibits HSC activation by directly targeting SMO.

Example 2. Confirmation of mitigation of liver fibrosis of MIR-486-5p

The direct action of miR-486-5p in mice with acute liver injury caused by CCl ₄ was investigated. Compared with mice treated with scrambled RNA (CCl ₄ +Scr group), the amount of AST/ALT decreased and hepatocyte function was decreased in mice injected with CCl ₄ and treated with miR-486-5p (CCl ₄ +miR-486-5p group). It was confirmed that liver damage was improved by increasing the level of G6pc, one of the markers (FIGS. 4A, 4B to 4C).

miR-486-5p was significantly upregulated, and Smo, Gli2 and the fibrotic markers α-Sma, Vimentin, CTGF and Col1α1 were significantly higher in the CCl ₄ + miR-486-5p group compared to the CCl ₄ +Scr group. It was confirmed that it was down-regulated (FIG. 4D).

Sirius red staining and IHC results for α-Sma clearly showed that liver fibrosis and HSC activation were reduced in the CCl ₄ +miR-486-5p group compared to the CCl ₄ +Scr group ( FIG. 4E ). These results suggest that miR-486-5p in hepatic fibrosis contributes to the alleviation of hepatic fibrosis by targeting Smo.

Taken together, these results suggest that miR-486-5p contributes to the alleviation of liver fibrosis by inhibiting the Hedgehog (Hh) pathway and inhibiting the activation of HSC.

As it was confirmed that miR-486-5p according to the present invention inactivates HSC by targeting Smo and suppressing Hh signaling, it was confirmed that miR-486-5p could be a new treatment for liver fibrosis.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Pusan National University Industry-University Cooperation Foundation <120> Pharmatheutical compositon comprising miR-486-5p for preventing or treating of hepatic fibrosis <130> ADP-2020-0644 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-486-5p <400> 1 gagccccguc gagucauguc cu 22 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 2 ttttctcgag ggggccatgt cctctcttaa 30 <210> 3 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer <400> 3 tgcggccgct tggaggctat ggaaggtgg 29 <210> 4 <211> 22 <212> RNA <213> Artificial Sequence <220> <223> miR-486-5p mimic <400> 4 uccuguacug agcugccccg ag 22

Claims (6)

A pharmaceutical composition for preventing or treating liver fibrosis, comprising miR-486-5p, an activator thereof or an expression promoter thereof as an active ingredient.
The method according to claim 1, wherein the miR-486-5p activator or expression promoter is any one selected from the group consisting of a compound that specifically binds to miR-486-5p, a peptide, an aptamer, and an antibody. , A pharmaceutical composition for preventing or treating liver fibrosis.
According to claim 1, wherein the miR-486-5p Hedgehog (Hedgehog, Hh) by inhibiting the expression of the receptor SMO in the pathway to inhibit the activation of hepatic stellate cells (Hepatic stellate cells, HSC) characterized in that, A pharmaceutical composition for preventing or treating liver fibrosis.
The pharmaceutical composition for preventing or treating liver fibrosis according to claim 1, wherein the liver fibrosis is non-viral liver fibrosis.
5. The method of claim 4, wherein the non-viral liver fibrosis is liver fibrosis, alcoholic cirrhosis, non-alcoholic cirrhosis, autoimmune cirrhosis, primary biliary cholangitis-associated cirrhosis, Wilson's disease-associated cirrhosis, hemochromatosis-associated cirrhosis, portal cirrhosis, postnecrosis. A pharmaceutical composition for preventing or treating liver fibrosis, characterized in that it is at least one selected from the group consisting of cirrhosis, nutrient deficiency-related liver cirrhosis, and cardiac cirrhosis.
A health functional food composition for improving liver fibrosis, comprising miR-486-5p, an activator thereof or an expression promoter thereof as an active ingredient.

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